Evaluation of postharvest storability of Ponkan mandarins stored at different temperatures

Fresh Ponkan mandarins grafted on rootstock trifoliate orange were harvested 195 days after full-bloom (DAF) stage with a ratio of TSS/TA >10 from a 16-year-old commercial orchard with the integrated management of water and fertiliser in the Xiangtian district of Jing’an City (28°48′47″ N and 115°23′59″ E, Jiangxi Province, China), in November 2018, and transported back to the Jiangxi provincial key laboratory of fruit and vegetable preservation and nondestructive testing (Nanchang, Jiangxi, China) within 4 h. After pre-storage for 3 days, the healthy fruits without diseases or mechanical damage and uniform in colour, size, shape and maturity were selected. Then, 1,800 fruits were randomly separated into four lots, and stored at 5 ± 1 °C (S5), 10 ± 1 °C (S10), 15 ± 1 °C (S15) and 20 ± 1 °C (Control) with a relative humidity of 85–90% for 120 days. We selected 15 fruits from each temperature lot every 15 days to evaluate their physiological and biochemical indexes.

The decay rate (DR) of Ponkan mandarins showing obvious symptoms of fungal disease was measured on the same 300 fruits of three replicates in each lot, and calculated on the initial fruit number for each lot every 15 days, and is expressed in percentage.

The weight loss (WL) of Ponkan mandarins was measured on the same 30 fruits of three replicates in each lot, and calculated on the initial fruit weight basis for each lot every 15 days, and is expressed in percentage of the initial weight.

The light (L*) value and citrus colour index (CCI) were measured using a MINOLTA CR-400 colorimeter (D65 light source; Konica Minolta Sensing, Inc., Osaka, Japan) for 15 fruits randomly from three replicates in each temperature lot, following the method of Rapisarda et al. (2001).

About 10.0 g of fruit pulp from five fruits per replicate in each temperature lot was homogenised and centrifuged at 7,500 × g for 15 min. The supernatant was obtained and used for measuring TSS content with the aid of a RA-250WE Brix-meter (Atago, Tokyo, Japan), and the result is expressed as °Brix. Both contents of TA and VC were determined by titration with 0.1 M sodium hydroxide (Chen et al., 2016) and 2,6-dichlorophenolindophenol (Rapisarda et al., 2001), which were expressed as a percentage of citric acid and mg · 100 g⁻¹, respectively. Both total phenol content (TPC) and total flavonoid content (TFC) were assayed based on the methods reported by Nie et al. (2020) using gallic acid (GA) and rutin as the standard, where the TPC and TFC were expressed as mg · g⁻¹ on the basis of frozen weight.

Fruit RI was measured using a GXH-3051H infrared carbon dioxide fruit and vegetable respiration tester (Jun-Fang-Li-Hua Technology-Research Institute Beijing, China) in units of mg CO₂ · kg⁻¹ · h⁻¹. We selected randomly five fruits per replicate in each temperature lot for measurement after weighting.

All experiments were performed in triplicate. All data were presented as means ± standard deviation (SD) with n = 3, and analysed by the Duncan's multiple range test at a significance level of 0.05 (p < 0.05) using SPSS 22.0 software (IBM SPSS 22.0, Chicago, IL, USA).

The DR and WL rate are important indexes for evaluating the storability of citrus fruit (Montero et al., 2009). Figure 1A illustrates the DR in all groups was kept to zero during the first 30 days of storage. The rotten fruit appeared in S5, S10, S15 treated, and control groups on the 45th day of storage. At the end of storage (120 days), the DR of S5 and S10-treated fruits were 40.2% and 52.1% lower than that of S20-treated fruits, respectively. We found significant differences between all groups (p < 0.05). Numerous previous studies have reported that low-temperature storage slows the DR of citrus (Henriod et al., 2005). Low-temperature storage may reduce the occurrence of fruit diseases and insect pests (Tietel et al., 2012).

Figure 1

Due to both water transpiration and nutrients degradation by respiration, the WL of all groups increased with the prolongation of storage. As shown in Figure 1B, the storage temperature at 5 °C and 10 °C could significantly reduce the WL of Ponkan mandarins (p < 0.05). At the end of storage, the lowest WL was 4.19% and was found in S5-treated fruit, which is 37.2% lower than that of the S20-treated (control) fruit. Some studies reported that, low-temperature storage can reduce fruit WL (Undurraga et al., 2009; Tietel et al., 2012), which may be due to the slow water transpiration and fruit respiration caused by low temperature, offering a relatively high humidity environment and maintaining the moisture and nutrients contents of fruits.

Low-temperature storage at a suitable temperature is beneficial for maintaining the appearance, colour and exterior quality of fruits, improving the storability, and prolonging the shelf life (Hong et al., 2013; Lado et al., 2018; Matsumoto et al., 2019). To some extent, the fruit colour reflects the freshness and postharvest quality of horticultural products. The L^* value decreased slightly with the prolongation of storage time (Figure 1C). During the whole storage period, the L^* value was significantly lower (p < 0.05) in S15 and S20 treated fruit compared with the S5 and S10 treated fruit. The CCI of Ponkan mandarins stored at different temperatures increased gradually with the increase in storage time (Figure 1D). However, under low-temperature storage, the increment of CCI in S5 and S10 treated fruit was significantly slower (p < 0.05) than that in S15 treated and control fruit. In this experiment, Ponkan mandarins maintained good appearance and lustre after long storage at 5 °C and 10 °C. These results are highly consistent with those reported in other studies (Matsumoto et al., 2009; Nawaz et al., 2020). Storage temperature affects the colour development and appearance quality trait in citrus fruit, thus significantly affecting its exterior quality. Ponkan mandarins stored at 10 °C have a better appearance, brighter L* value, lower CCI and more sluggish fruit senescence. These results also indicate that too low storage temperature (5 °C) is not conducive for fruit colouration and after-ripening.

The TSS, TA and VC contents are important factors for assessing the nutritional quality and flavour of citrus fruits (Lado et al., 2018; Chen et al., 2019). TSS in citrus juice is composed of acid soluble pectins, vitamins, sugars and some soluble proteins (Alhassan et al., 2019). The TSS content of fruits stored at different temperatures increased first to achieve their peaks, followed by a decline in TSS content during the remainder of the storage period. The peak of TSS content in each treatment group differed, and then the TSS content of each treated fruit decreased rapidly (Table 1A). The TSS content of S10-treated fruit was significantly higher (p < 0.05) than the other three treatments during the mid–late-storage periods (60–120 days). Similar results were reported by Alhassan et al. (2019) for Afourer mandarins and navel oranges.

The TA content is regarded as an important index for evaluating fruit flavour and RR of horticultural crops (Bal, 2013; Chen et al., 2019). In our study, during storage, the TA content in all groups decreased continuously with the increase of storage time, with the control group undergoing the fastest degradation of TA. At the end of storage (120 days), the TA content in S5, S10, S15 and S20-treated groups were 0.63%, 0.61%, 0.38% and 0.33%, respectively (Table 1B). The TA content of S5 and S10-treated fruit retained the initial level of 38.7% and 37.4%, whereas only 20.2% was retained in the S20-treated fruit. In this study, the high TA content in the S10-treated fruit may be due to the lower RR, which delayed the degradation of TA (Tietel et al., 2012; Alhassan et al., 2019).

Vitamin C is one of the key factors used to evaluate the quality of mandarin fruit. As shown in Table 1C, the VC content of fruits stored at different temperatures first increased and then decreased in the subsequent storage period. The peak times of VC content in each treatment group differed (e.g. 15 days in both S15 and S20 groups and 30 days in both S5 and S10 groups). The VC content in the S10-treated fruit was significantly higher than the other three treatments after the 15th day of storage (p < 0.05). These results are similar to those reported by Baltazari et al. (2020); the storage temperature was found to be an important factor affecting the nutritional quality of citrus. Therefore, determining the best minimum safe storage temperature for each citrus variety is important.

Phenols and flavonoids are important secondary metabolites in plants. Most of them have the capacity to scavenge free radicals and play an important role in the plant defence mechanism (Nie et al., 2020; Piljac-Zegarac and Samec, 2011). Both contents of total phenols and flavonoids in Ponkan mandarins increased first, and then decreased during the remainder of storage (Table 1). The TPC reached a peak at 45 d and then decreased (Table 1D). The decrease in TPC in S10-treated fruit was significantly lower than that of the other three treatments after the 45th day of storage (p < 0.05). The changes in TFC were likely similar to that of TPC, reaching the peak at the 60th day of storage and then decreasing in the subsequent storage period. The TFC in S10-treated fruit was 1.49 times higher than that at the beginning of storage (Table 1E), and was significantly (p < 0.05) different from that of the other three treatments. In this study, high TPC and TFC were maintained in S10-treated fruit (Table 1), consistent with a previous report that low-temperature storage can maintain high anthocyanins content in blood orange (Rapisarda et al., 2001). In this study, the results indicated that the appropriate storage temperature could improve the oxidation resistance of fruits by increasing the secondary metabolites with defensive ability in fruit tissues.

The RR of Ponkan mandarins increased under different temperature treatments (Figure 2A). Low-temperature storage significantly slowed the increase in the RI of Ponkan mandarins, and a significant difference (p < 0.05) in both S5 and S10-treated groups was found between the earlier and later stage of storage compared with the S15 and S20-treated groups (p < 0.05). Similar results were reported by Koh et al. (1998) for Satsuma mandarin storage; the RI of Satsuma mandarin fruit storage at 10 °C was significantly lower than that at the other three temperatures (4 °C, 20 °C and 35 °C) treatments. This is analogous to low-temperature storage, which can reduce fruit respiration of Ponkan mandarins during storage.

Figure 2

The level in REC of Ponkan mandarins increased irrespective of storage temperature (Figure 2B), because the gradual senescence of fruits during storage may lead to increased permeability of the pericarp cell membranes. After storage for 120 days, the REC was 32.6% in S5-treated fruit, and 30.1% in S10-treated fruit, 36.2% in S15-treated fruit and 39.2% in S20-treated fruit, respectively. The REC in S10-treated fruit was significantly lower (p < 0.05) than in the other three treatments, suggesting that too low storage temperature might lead to chilling damage, and high storage temperature may accelerate the nutrients degradation and fruit senescence.

Hydrogen peroxide is one of the important representatives of reactive oxygen species (ROS), and its accumulation causes fruit senescence (Mittler, 2002). The extension of storage time promoted the H₂O₂ accumulation; after the peak of H₂O₂ content at 75 days of postharvest storage, it rapidly decreased (Figure 2C). In comparison with the other three treatments, the H₂O₂ content slowly increased and then quickly decreased, suggesting that low-storage temperature inhibits the H₂O₂ accumulation and exhibited a lower level of H₂O₂ content, compared with that observed in those fruits stored at too low and high storage temperature (S5, S15 and S20 groups; p < 0.05). In this study, low-temperature storage delayed fruit senescence, resulting in a reduction in the accumulation of H₂O₂.

The damage of oxidative stress increased during storage, and excess ROS increased the MDA accumulation and destroyed cell membrane structure, thereby accelerating fruit senescence (Chen et al., 2019; Mittler, 2002). Membrane lipid peroxidation was expressed as MDA content (the final decomposing product of membrane lipid peroxidation), which is closely related to ageing and is one of the direct indicators of membrane oxidative damage. Figure 2D shows that the MDA content in each treatment presented an escalating tendency as the storage time increased. Fruit stored at 10 °C had the lowest MDA content compared with the other three treatments after storage (p < 0.05; Figure 2D). Therefore, the accumulation of MDA may be related to the senescence and high VC content of fruits stored at low temperature. To further understand the reason for this change, the future analysis protective enzyme activity that delays lipid peroxidation and cell ageing is needed.

The activities of SOD, CAT, POD and APX are closely related to antioxidation and anti-ageing in plant tissues (Rao et al., 2011; Nie et al., 2020). During the entire storage period, these enzyme activities in Ponkan mandarins initially increased and then notably decreased (Figure 3). As revealed in Figure 3A, the SOD activity in control, S15, S10 and S5-treated fruit reached their highest levels at 45 days and 60 days, indicating that low storage temperature can effectively improve SOD activity and retard the peak time during the storage time. The higher level of SOD activity was noticed in S10-treated fruit compared with the other three treatments (S5, S15 and S20) at various sampling times (p < 0.05; Figure 3A). The CAT activity levels in S10-treated fruit reached a maximum value on the 45th day, exhibiting values that were 32.89%, 8.56% and 40.14% higher relative to its levels in S5, S15 and S20 fruit, respectively (Figure 3B). The POD activity peaked at 30 days and then decreased rapidly (Figure 3C). The decline of POD activity in S10-treated fruit was significantly slower (p < 0.05) than the S5, S15 and S20 groups. A significantly elevated APX level was observed in Ponkan mandarins exposed to low temperature compared with the S20-treated fruit throughout the storage (Figure 3D). From an initial storage (0 days) to the end storage, the increase in APX activity in S5, S10 and S15-treated fruit was on average 114.1%, 66.68% and 32.97% higher than that in the S20-treated fruit, respectively (p < 0.05).

Figure 3

The excessive production and accumulation of ROS caused by fruit senescence destroys the integrity of the cell membrane and reduces the storage tolerance of fruit (Mittler, 2002; Undurraga et al., 2009). The high activity of antioxidant and defence-related enzymes can effectively reduce the accumulation of ROS and MDA, reduce oxidative damage, delay fruit ageing and prolong storage life. As important antioxidant enzymes, SOD and POD can prevent membrane lipid peroxidation caused by excessive ROS (Gill and Tuteja, 2010; Rao et al., 2011). CAT plays an important part in the scavenging process of ROS. As a stress reaction, CAT usually changes with the change in ROS level. APX is the key enzyme for removing H₂O₂ in chloroplasts and the main enzyme for VC metabolism. In our study, the results indicated that low-temperature storage enhanced the activities of SOD, CAT, POD and APX, and lessened the accumulation of MDA (Figure 2 and 3). These results are consistent with those of Chen et al. (2019) and Zhang et al. (2011), compared with room temperature storage, where two citrus fruit varieties of Xinyu tangerines and ‘Guanximiyou’ pummelos stored at 6 °C exhibited the increase in antioxidant enzyme like SOD, CAT and POD activities, thereby inhibiting MDA accumulation and enhancing postharvest quality storage life.

A PCA analysis was performed to assess the preservation efficacy of different storage temperatures on the postharvest quality of Ponkan mandarin by evaluating 17 physicochemical parameters. The eigenvalues of the covariance matrix suggested that the two principal components (PCs) are able to account for 87.233% of the total variance in the dataset, and PC1 was found to explain 58.195% and the PC2 explained an additional 29.038% of the variance (Figure 4A). The absolute value of the eigenvalues mirrored the contribution rate to each PC, and the higher the absolute value, the greater the contribution rate. As illustrated in Figure 4B, both positive and negative correlations were intuitively observed among 17 postharvest physicochemical parameters. Interestingly, the seven positive axis of PC1 were decay rate, weight loss, CCI, REC, H₂O₂ content, RI and MDA content. Conversely, PC1 was negatively correlated with L* value, POD activity and the contents of TSS, TA, VC (ascorbic acid) and total phenols. PC2 had high positive loading for TFC, SOD, CAT and APX activities (Figure 4B). Notably, the preservable index (e.g. decay rate and weight loss) of Ponkan mandarin was significantly positively correlated with CCI, RI, REC, H₂O₂ content and MDA content (p < 0.01), and negatively correlated with L* value, the contents of TSS, TA, VC and total phenols, and POD activity (p < 0.01), respectively. Figure 4B shows that the comprehensive score of Ponkan mandarin increased gradually with storage time. Both the S5 and S10-treated fruit showed a lower increase in comprehensive score compared with the S20-treated and S15-treated fruit during the whole storage period. No significant difference was found in the comprehensive score between the S5 and S10-treated fruit after the 30th day of storage until the end of storage (Figure 4C). The PCA results showed that Ponkan mandarin stored at 10 °C exhibited better preservation effect compared with the control group. Postharvest low temperature storage treatment was effective to preserve Ponkan mandarin quality during the storage at 10 °C. PCA is an effective way to evaluate the postharvest storability of different storage temperatures on Ponkan mandarin as reported by other studies in several citrus fruit, such as kumquat (Liu et al., 2018), ‘Newhall’ navel orange (Wan et al., 2020), and ‘Majiayou’ pummelo (Nie et al., 2020).

Figure 4